Intramuscular Injection of AAV8 in Mice and Macaques Is ......Intramuscular Injection of AAV8 in...
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Intramuscular Injection of AAV8 in Mice and Macaques IsAssociated with Substantial Hepatic Targeting andTransgene ExpressionJenny A. Greig, Hui Peng, Jason Ohlstein, C. Angelica Medina-Jaszek, Omua Ahonkhai, Anne Mentzinger,
Rebecca L. Grant, Soumitra Roy, Shu-Jen Chen, Peter Bell, Anna P. Tretiakova, James M. Wilson*
Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, TRL Suite 2000, 125 South 31st
Street, Philadelphia, PA, 19104, United States of America
Abstract
Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinicaldevelopment with some success, including the first approved gene therapy product in the West called Glybera. Inpreparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies inmice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administrationof AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly tototal expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and bythe use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements.Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAVdelivery should inform safe and efficient development of future AAV products.
Citation: Greig JA, Peng H, Ohlstein J, Medina-Jaszek CA, Ahonkhai O, et al. (2014) Intramuscular Injection of AAV8 in Mice and Macaques Is Associated withSubstantial Hepatic Targeting and Transgene Expression. PLoS ONE 9(11): e112268. doi:10.1371/journal.pone.0112268
Editor: Knut Stieger, Justus-Liebig-University Giessen, Germany
Received March 31, 2014; Accepted October 6, 2014; Published November 13, 2014
Copyright: � 2014 Greig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: This work was supported by the Bill & Melinda Gates Foundation, grant number 51061 and URL http://www.gatesfoundation.org/. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: J.M. Wilson is an advisor to ReGenX Biosciences and Dimension Therapeutics, and is a founder of, holds equity in, and receives grantsfrom ReGenX Biosciences and Dimension Therapeutics; in addition, he is a consultant to several biopharmaceutical companies and is an inventor on patentslicensed to various biopharmaceutical companies. Numerous patents describing the isolation of various AAVs and their use in gene therapy have originated fromthe Gene Therapy Program. Several patents and applications describe methods of producing AAV vectors. Some patents have issued, and some are underprosecution. Our portfolio relating to AAV vectors spans over 200 patents and applications worldwide. This does not alter the authors’ adherence to PLOS ONEpolicies on sharing data and materials. All other authors declare no competing interests.
* Email: [email protected]
Introduction
Vectors derived from adeno-associated viruses (AAV) have been
shown to produce long-term and stable gene expression of secreted
proteins in a variety of animal models and human clinical trials
following intramuscular (IM) injection, including coagulation
factor IX (FIX) [1,2,3,4,5], alpha-1-antitrypsin (AAT) [6,7],
erythropoietin [8], and neutralizing immunoglobulins against
HIV [9,10]. Intramuscular (IM) delivery of an AAV vector
provides a quick, easy, non-invasive and safe route of administra-
tion, which can be routinely performed in virtually any setting.
The most celebrated example of IM AAV gene therapy is the
treatment of an inherited deficiency of lipoprotein lipase with the
commercially approved product Glybera [11]. However, previous
studies have identified some of the limitations of IM injections,
whereby transduction is limited to cells around the needle tract
area of the injection site in mice, nonhuman primates (NHP) and
humans [3,12,13,14,15]. This has led to the practice of a large
number of small volume IM injections to produce sufficient
transgene expression [7]. For example, subjects enrolled in the
high dose cohorts of the Phase II AAT clinical trial received one
hundred 1.35 ml vector injections IM spread across ten sites or up
to sixty 0.5 ml injections in clinical trials for Glybera [6,11].
Local injection of vector into most tissues could lead to a
percentage of the injected volume disseminating from the site of
injection and being transported to other organs. We speculate that
the larger the volume of an IM injection, potentially the greater
fraction of the volume can be dispersed from the site of
administration. Therefore, due to the natural or increased tropism
of certain AAV vectors for the liver and the resulting liver
transduction, a significant contribution to the total level of a
secreted transgene protein may be contributed by the liver
following IM administration. Also, distribution of vector beyond
the muscle could have implications on the safety and immuno-
genicity of the treatment. It has been suggested that delivery of the
vector to liver, either by design or inadvertently, could induce
immunologic tolerance to the transgene product, thereby dimin-
ishing immune toxicity [16,17,18,19,20,21,22,23,24]. In contrast,
IM administration of concentrated AAV vectors, resulting in high
vector dose per injection site, has been linked to higher level
antibody production against the secreted transgene product [4,5].
An AAV product can be engineered to restrict the expression of
transgenes following different systemic routes of administration,
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such as IM, which will lead to broad distribution of vector. The
more traditional approach to overcome this problem is to drive
expression of the transgene from a tissue-specific promoter, such as
the muscle creatine kinase (tMCK) promoter for skeletal muscle
expression [25] and the human thyroxine binding globulin (TBG)
promoter for liver expression [26,27]. Transgene expression can
also be inhibited in certain organs by the incorporation of tissue-
specific microRNA target sites [28,29]. Interaction of microRNAs
with their complementary target sites within the RNA-induced
silencing complex can lead to inhibition of translation or
degradation of mRNA [30,31]. Incorporation of 3–6 copies of
target sites for the liver-specific microRNA (miR) 122 or skeletal
muscle-specific miR-206 in the 39 UTR of an AAV vector has
been previously shown to reduce liver and muscle gene expression,
respectively [32,33,34,35]. Therefore, transgene expression could
be restricted from either liver by miR-122 or muscle by miR-206.
In the current study we have evaluated important aspects of
AAV8 IM delivery, such as concentration and volume, and
features of the expression cassette, such as tissue specificity, on
safety and efficacy in mice and macaques.
Results
Substantial gene expression from liver following IMadministration of AAV8 vectors in mice
AAV8 vector expressing firefly luciferase (ffLuc) from the
ubiquitous CMV promoter was injected IM into C57BL/6 mice at
a dose of 1010 genome copies (GC) per mouse (Figure 1A). Vector
was administered as one 10 ml injection into the right gastrocne-
mius muscle and on day 7 post-vector mice were imaged to
determine the localization of ffLuc expression. Significant ffLuc
expression, which localized to both the injected muscle and the
liver, was demonstrated (Figure 1A). These imaging results were
quantitatively reproduced and further expanded using biochemical
assays where ffLuc was measured in both liver and muscle samples
and normalized to total protein on day 28 post-vector adminis-
tration (Figure 1C). IM injection of vector using the CMV
promoter for expression resulted in extensive ffLuc expression in
the injected muscle with concordant expression in the liver, which
was 321-fold over background (control un-injected mice).
The unexpectedly high levels of liver transduction after IM
injection suggested that IM delivery could be an alternative to the
standard way of targeting liver, which is by intravenous (IV)
injection. Experiments were repeated with a vector expressing
ffLuc from the highly potent, liver-specific promoter TBG [26,27].
Mice were injected IM with 1010 GC of AAV8.TBG.ffLuc in a
volume of 10 ml and at day 7 post-vector administration
significantly higher liver expression was seen with little to no gene
expression in muscle (Figure 1B). ffLuc tissue assays on liver and
muscle taken at day 28 following administration of vector showed
an increase in liver ffLuc expression of 69-fold, relative to that
achieved with the CMV promoter (Figure 1C). While muscle
ffLuc expression was over background (control un-injected mice)
following IM injection, the three log reduction in muscle
expression seen was expected due to the specificity of the TBG
promoter (Figure 1C).
As ffLuc expression allowed tissue localization of gene
expression, the net impact of inadvertent liver delivery of AAV
following IM injection was studied by expression of a secreted
protein. As antibodies expressed from AAV are being developed to
prevent infections, including HIV and influenza [9,10,36,37],
these initial studies used a gene encoding 201Ig IA. This
immunoadhesin (IA) construct was based on the 201Ig FAb,
which was isolated from a long-term non-progressing rhesus
macaque six years post-challenge with SIVsmF236 [38,39]. RAG
KO mice were injected IM with 1010 GC of AAV8 expressing
201Ig IA from the CMV or TBG promoters (Figure 1D).
Figure 1. Liver expression following IM vector administrationin mice. Visualization of differential ffLuc expression patterns usingXenogen whole-body bioluminescent imaging on day 7 post-IMadministration of 1010 GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B)to C57BL/6 mice. Vector was administered as one 10 ml injection intothe gastrocnemius muscle of the right leg. (C) ffLuc expression wasquantified at day 28 post-vector administration by ffLuc tissue assays.ffLuc expression measured as RLU normalized to the total proteinconcentration of the organ. Data are presented as fold change overbackground where background was RLU/total protein of organ (g) incontrol tissues from un-injected mice. (D) Comparison of expression of201Ig IA from the CMV and TBG promoters following a 10 ml IMinjection in RAG KO mice. Expression of 201Ig IA in serum wasmeasured by ELISA. Values are expressed as mean 6 SEM (n = 4/group).***p,0.001.doi:10.1371/journal.pone.0112268.g001
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Immunodeficient RAG KO mice were used to evaluate expression
of the 201Ig IA transgene in the absence of an immune response to
the secreted protein. Substantial levels of IA expression were
detected in blood with both vectors, although the liver-specific
promoter yielded an almost 8-fold higher expression of the
secreted protein in blood. This suggests that a substantial amount
of the secreted IA is derived from liver, rather than muscle, after
IM injection. Additional studies to evaluate the relative contribu-
tion of the two tissues/organs to blood levels of the transgene
product are described below.
Dose and route of administration impacts on expressionin mice
AAV8 vectors expressing anti-SIV/SHIV antibodies (in an
immunoadhesin format [i.e., 201Ig IA] or a monoclonal antibody
format [i.e., 2.10A mAb] [40]) or human anti-HIV antibodies in a
monoclonal antibody format (i.e., VRC01 mAb [41,42] and PG9
mAb [43]) were injected IM as two 15 ml injections at doses of 1010
GC or 1011 GC into RAG KO mice (Figure 2). Expression of
these proteins in serum was determined on day 28 post-vector
administration and compared to expression from TBG-containing
vectors administered intravenously (IV) via the tail vein. Figure 2
summarizes data from these experiments, in which the two doses
of vectors (labeled 0.1 and 1) were evaluated in the context of two
comparisons: the CMV versus TBG vectors following IM injection
and the TBG vector following IM and IV injection.
A dose-dependent increase in transgene expression was seen for
all AAV8 vectors, independent of route of administration or
promoter, where expression increased on average 8.5-fold across
all transgenes when vector dose per mouse was increased by one
log (Figure 2). Substantial differences were achieved when
comparing expression from the CMV versus TBG promoter of
vectors administered IM. A statistically significant increase in
expression was observed with the TBG promoters, compared to
the CMV promoters, at the highest dose of vector with the three
mAb cassettes (Figures 2B-D). A direct comparison of IM versus
IV injection of the TBG promoted vectors revealed little difference
in expression of the antibodies at either dose of vector (Figures 2B-
D). The exception to this was 201Ig IA, which was significantly
increased (4.8-fold) following IV injection (Figure 2A). Therefore,
a comparable level of secreted gene expression from a liver-specific
promoter can be produced following a quick, easy and non-
invasive injection into skeletal muscle or an invasive IV injection,
which requires a higher level of skill.
Modulation of liver and muscle gene expression by IMinjection volume in mice
We investigated the impact of injection volume on the level and
distribution of transgene expression. In these studies the same dose
of vector was injected in a range of volumes from two 25 ml
injections, one into each leg for a total injection volume of 50 ml, to
one 2 ml injection in one site representing a 25-fold range of vector
concentrations. The initial studies focused on mice injected with
Figure 2. Comparison of expression from the liver-specific TBG promoter following IM and IV vector administration in RAG KOmice. Expression on day 28 post-vector administration of 201Ig IA (A), 2.10A mAb (B), VRC01 mAb (C), and PG9 mAb (D) in RAG KO mice. Expressionfollowing IM injection of vectors with either the CMV or TBG promoter was compared to expression levels produced following IV injection of the TBGvector. Mice were injected IM with two 15 ml injections into the right and left gastrocnemius muscles. IV injections were performed as a 100 mlinjection via the tail vein. Mice were administered with a dose of either 1010 GC or 1011 GC, numbers indicate dose 61011 GC. ND, not determined.Expression was measured in serum by ELISA and values are expressed as mean 6 SEM (n = 3/group). ***p,0.001.doi:10.1371/journal.pone.0112268.g002
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1010 GC AAV8 vectors expressing ffLuc from the CMV or TBG
promoters. Tissues were harvested at day 28 and transgene
expression measured in lysates from liver (Figure 3A) and muscle
(Figure 3B). Our original hypothesis was that increasing the
volume for a fixed dose would increase the relative distribution of
vector to liver. These studies did confirm the pilot experiments
described in Figure 1, which used a single concentration of vector,
whereby: 1) IM injection of vector resulted in substantial levels of
transgene expression in liver, and 2) vectors using the TBG
promoter produced levels of transgene product in liver following
IM injection that were almost equivalent to the levels achieved
when the same vector was injected IV. However, we were
surprised to see that the more concentrated IM injections of vector
did not help restrict expression to muscle; in fact these studies
showed higher levels of liver ffLuc expression with lower volumes
of vector. Also, the higher concentrations of vector yielded higher
levels of overall ffLuc in both muscle and liver, independent of
promoter. Based on in vitro experiments (data not shown), there
was no significant loss of vector following dilution to different
concentrations prior to administration in mice. Therefore, all mice
received the same dose of vector and differences in distribution of
the vector were due to the concentration of the injected vector.
The impact of IM injection volume was also studied in mice
injected with AAV8 vectors expressing 201Ig IA from either the
CMV (Figure 3C) or TBG promoter (Figure 3D) with the readout
being serum levels of 201Ig IA. The CMV vectors (Figure 3C)
showed dramatically lower overall expression as compared to the
TBG vectors (Figure 3D). Serum 201Ig IA levels were not
markedly affected by the volume of vector injected IM. Although
the highest expression from the IM injected TBG vector was
achieved with the lowest volume, it was still around two-fold lower
than that achieved following IV injection.
A series of studies were performed using LacZ as a reporter gene
to evaluate distribution of transduction at a cellular level as a
function of vector concentration and dose. C57BL/6 mice were
injected IM with AAV8 expressing LacZ from the CMV
promoter. Liver and muscle tissue were harvested for analysis by
LacZ histochemical staining 21 days post-vector administration
(Figure 4). Sections of the gastrocnemius muscles were taken at
intervals throughout the entire injected muscle. At a dose of 1010
GC, IM injection of vector as either two 25 ml injections
(Figure 4A) or one 2 ml injection (Figure 4C) produced similar
patterns of expression throughout the injected muscle, with a few
transduced cells being seen in the liver (Figures 4B, 4D); note that
the CMV promoter does not express well in liver. To determine if
gene expression was saturated in the muscle at a dose of 1010 GC
per mouse, the dose was lowered to 109 GC per mouse and a very
different transduction pattern was seen for the two injection
volumes (Figures 4E, 4G). Injection of the vector IM as two 25 ml
injections produced few transduced cells in the muscle (Figure 4E).
Figure 3. Reduction in IM injection volume increases transgene expression. 1010 GC AAV8 expressing ffLuc from the CMV or TBG promoterwas delivered IM as either two 25 ml injections (one into each leg), one 10 ml injection or one 2 ml injection to C57BL/6 mice. IV injections wereperformed as a 100 ml injection via the tail vein. ffLuc tissue assays were performed on tissue harvested at day 28 and normalized to the total proteinconcentration of liver (A) and muscle (B). RAG KO mice were administered with 1010 GC AAV8.CMV.201Ig IA (C) or AAV8.TBG.201Ig IA (D) by IV or IMinjections, performed as described previously. Data for the one 10 ml injection groups were previously presented in Figure 1. Expression of 201Ig IA inserum was measured by ELISA. Values are expressed as mean 6 SEM (n = 4/group). *p,0.05, **p,0.01, ***p,0.001.doi:10.1371/journal.pone.0112268.g003
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When the concentration of the vector was increased to enable
injection of the same dose of vector in a volume of 2 ml, a large
area of transduced skeletal muscle cells were seen (Figure 4G).
This transduced area extended through multiple muscle sections.
Again, only a few transduced cells were seen in the liver at a vector
dose of 109 GC per mouse (Figures 4F, 4H). Therefore,
administration of the same vector dose as a smaller injection
volume improved skeletal muscle transduction at lower overall
vector doses.
Contributions of liver and muscle to total expression of asecreted transgene in mice
A critical issue not addressed in the experiments described
above is the relative contribution of liver versus muscle in the
production of secreted transgene products following IM injection
of AAV8 vectors. Additional studies focused on vectors expressing
the antibody 201Ig IA from a CMV promoter to simulate the
likely clinical application of AAV expressed antibodies for
prevention of infections, such as HIV and influenza
[9,10,36,37]. The strategy employed to address this issue utilized
microRNA target sites that allow for tissue specific ablation of
transgene expression. For example, the microRNA target sites for
the liver-specific and the skeletal muscle-specific microRNAs,
miR-122 and 206, respectively, have been previously shown to
reduce gene expression in liver and muscle following the
incorporation of these target sites within an AAV vector genome
[32,33,34,35]. Initial studies were performed with AAV8 vectors
expressing ffLuc from a CMV promoter, with and without miR-
122 and miR-206 target sites, in order to confirm the activity of
the microRNA target sites. C57BL/6 mice injected IM with the
ffLuc vectors were imaged for ffLuc expression on day 7
(Figures 5A-C) and expression was quantified on day 28
(Figure 5D). Incorporation of target sites for miR-122 and miR-
206 into the 39 UTR of the transgene produced specific
knockdown of gene expression in the liver and muscle of 6.6-
and 112-fold, respectively (Figures 5A-D). As previously demon-
strated [32,33,35,44,45,46], expression of the transgene in the
organ that was not the target of microRNA-induced knockdown of
gene expression remained unchanged (Figures 5A-D). These
studies confirmed the usefulness of these microRNA target sites
in specifically ablating liver or muscle expression.
Studies were conducted with vectors in which ffLuc was
substituted with 201Ig IA in RAG KO mice, to evaluate
expression in the absence of an immune response to the secreted
protein. Expression of 201Ig IA following IM administration of an
AAV8 vector containing the CMV promoter in the absence of
miRNA target sites plateaued at 410 mg/ml on day 42 post-vector
administration (Figure 5E). Restriction of transgene expression to
muscle by the incorporation of miR-122 target sites reduced 201Ig
IA expression, although not significantly, to 366 mg/ml (Fig-
ure 5E). In the presence of target sites for miR-206 to restrict
expression to liver, 201Ig IA expression was significantly reduced
to 169.2 mg/ml compared to the control vector (Figure 5E). GC
analysis was performed for all groups and there was no significant
difference in the number of GC present in the liver at day 90 post-
vector administration, suggesting that the reduction in expression
was due to the presence of the miRNA target sites and not
ineffective vector administration (Figure 5F). These studies indi-
cate that expression of a secreted antibody following IM injection
of a CMV driven AAV8 vector in mice is primarily derived from
muscle, although liver does contribute a substantial amount to
total transgene expression.
Hepatic distribution of AAV8 vector following IMinjection is similar between mice and macaques
To help evaluate the relevance of these observations in mice to
human clinical trials, we conducted comparison studies with
rhesus macaques. In doing so we could not perform parallel studies
Figure 4. Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice. Localization of LacZexpression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 1010 GC astwo 25 ml injections into the right and left legs (A, B) or as one 2 ml injection (C, D). A lower dose of 109 GC of vector was administered as two 25 mlinjections into the right and left legs (E, F) or as one 2 ml injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle ofone representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).doi:10.1371/journal.pone.0112268.g004
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of luciferase imaging since the size of the macaques precluded the
use of this imaging modality. Therefore, we restricted our
comparison studies to that of vector genome biodistribution.
Mouse studies were performed with an AAV8 vector expressing
201Ig IA from a CMV promoter with tissues harvested at day 56.
Animals were injected IM with 1010 GC of vector in different
volumes (2625 ml, 1610 ml, and 162 ml). The deposition of vector
significantly increased in liver as the concentration of the vector
increased, consistent with the effects of vector concentration on
transgene expression (Figure 6A). While there was not a significant
increase in muscle, there was a trend towards higher GC following
administration of smaller injection volumes (Figure 6B). The
amount of vector present in the liver from the most concentrated
dose delivered IM was close to, but slightly lower, than that
observed following an IV injection of the same dose of vector
(Figure 6A). Injection of vector IM in its most concentrated form
yielded GC/diploid genome in muscle 2-fold lower than that in
liver, again supporting substantial targeting of liver after IM
injection of vector.
A male rhesus macaque was administered with the same vector
at a dose of 361012 GC/kg by IM injection into the right and left
vastus lateralis muscles, as 1 ml injections per kg body weight. This
simulates the conditions of vector delivery in the mice in terms of
concentration (NHP = 361012 GC/ml; mice = 561012 GC/ml
with 2 ml injection and 161012 GC/ml with 10 ml injection),
although the total dose of vector administered to mice was 10-fold
lower than in NHPs. Figure 6C presents a biodistribution analysis
of vector across a wide range of tissues, including muscle and liver.
GC in the injected muscles are presented as the average of samples
taken from 12 sampling sites within the muscle, which average 7.3
and 6.6 GC/diploid genome for the right and left injected
muscles, respectively (Figure 6C). No GCs were detected in the
control (un-injected bicep) muscle samples. GC for the four lobes
of the liver were determined separately, the average of which was
7.8 GC/diploid genome (Figure 6C). GC biodistribution in the
rhesus macaque were not qualitatively different from that in mice.
GC in muscle and liver was higher in the macaque than in mice
presumably reflecting the 10-fold higher dose of vector adminis-
tered to the macaque, although the increase was higher in muscle
(3-fold) than what was observed in liver (50%). 201Ig IA RNA
levels in the injected muscles and liver were also determined
(Figure 6C). Very high level transcription of the transgene was
seen in the injected muscles with substantially lower corrected
relative expression of 201Ig IA per GC seen in liver, possibly due
to the reduced activity of the CMV promoter in liver. Serum levels
of 201Ig IA were determined in the injected rhesus macaque and
Figure 5. Effect of microRNA target sites on localization and expression level following IM injection of AAV8 in mice. Differential ffLucexpression patterns were visualized on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A), AAV8.CMV.ffLuc.miR-122 (B) orAAV8.CMV.ffLuc.miR-206 (C). ffLuc expressing vectors were administered to C57BL/6 mice in two 10 ml injections into the right and leftgastrocnemius muscles. (D) Liver and muscle expression of ffLuc was quantified separately on day 28 post-vector administration. Followingsubstitution of ffLuc for 201Ig IA, expression of 201Ig IA was determined in RAG KO mice. Mice were injected with 1011 GC of vectors, administered asdescribed previously and expression of 201Ig IA in serum was measured by ELISA (E). GC of the 201Ig IA vectors present in the liver of RAG KO mice atday 90 post-vector administration was determined (F). Values are expressed as mean 6 SEM (n = 3/group). *p,0.05, ***p,0.001.doi:10.1371/journal.pone.0112268.g005
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no drop in transgene expression was seen throughout the course of
the study, indicating a lack of either neutralizing antibody (NAb)
production to the transgene or destructive cytotoxic T lymphocyte
response to the transgene expressing muscle (Figure 6D).
Small volume vector administration by IM injection hasno effect on transgene expression in rhesus macaques
To allow for translation of the effect of vector concentration on
expression in mice to human subjects in gene therapy clinical
trials, a large animal model was required for extrapolation of more
relevant vector doses per injection site and per kg body weight.
Rhesus macaques were administered IM with 361011 GC/kg of
AAV8.CMV.201Ig IA with the vector being injected as either
1 ml vector injections per kg body weight (361011 GC/ml) or
0.1 ml per kg body weight (361012 GC/ml). There was no
significant difference in 201Ig IA expression between the groups,
which has remained constant for nearly one year post-vector
administration (Figure 6E). Contrary to previous observations, for
AAV8 vectors expressing 201Ig IA in rhesus macaques there
appears to be no evidence of NAbs to the transgene being
generated with high vector doses per injection site.
Discussion
An important finding from our study is the extensive
distribution of AAV8 vector to liver following IM injection in
both mice and macaques. While this issue has indeed been raised
as a consequence of muscle delivery in previous studies, we show
that the hepatic deposited gene may contribute substantially to
systemic production of transgene products, especially if the
promoter is active in hepatocytes. These findings have relevance
to both pre-clinical and clinical applications of gene therapy.
A number of studies have attempted to evaluate the perfor-
mance of AAV-mediated gene transfer to skeletal muscle in the
context of secreted transgene products. We show, using micro-
RNA target sites to selectively ablate liver versus muscle
expression, that with AAV8 vectors containing the more muscle-
specific CMV promoter, the majority of transgene product in the
blood is derived from transduced muscle with liver contributing to
at least one third of the total product. Liver dominated the
production of the secreted transgene product following IM
injections with a vector containing a potent and liver-specific
promoter. Therefore, one may erroneously ascribe vector perfor-
mance to muscle transduction following IM injection in situations
with capsids that target liver and with promoters that have activity
Figure 6. Translation of influence of injection volume to a large animal model, the rhesus macaque. RAG KO mice were injected with1010 GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified asGC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesusmacaque. Vector was administered at a dose of 361012 GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 mlinjections per kg body weight (vector concentration of 361012 GC/ml). DNA and RNA were extracted for quantification of GC (open bars) andtranscript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscleand liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) musclesamples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E)Rhesus macaques were injected IM with 361011 GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (361011 GC/ml) or0.1 ml injection per kg body weight (361012 GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressedas mean 6 SEM. *p,0.05.doi:10.1371/journal.pone.0112268.g006
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in both muscle and liver, such as the frequently used CMV-
enhanced b-actin (CAG) promoter.
The inadvertent targeting of liver following IM injection of
skeletal or cardiac muscle has been seen as a safety concern and
has led to strategies to ‘‘de-target’’ liver through capsid engineer-
ing or restricting transcription. A series of studies have emerged
that suggest some delivery of vector to the liver may actually be
beneficial in terms of avoiding immune toxicity. Herzog and
colleagues were the first to show that IV delivery of AAV was
associated with induction of tolerance via the activation of
transgene product specific regulatory T cells, which is consistent
with the previously known tolerogenic nature of the liver
[16,17,18,19,20,21,22,23,24]. AAV induced tolerance is being
considered as a way to avoid immune responses in protein
replacement therapies. The properties of the AAV capsid will also
influence transgene immunogenicity. We previously showed in
hemophilia B mice that IM injection of hepatotropic vectors, such
as AAV8, avoided factor IX antibodies, which was not the case
with vectors that poorly transduce liver, such as AAV1 and AAV5
[47].
Our studies also demonstrate the impact of vector dose and
concentration on efficiency of muscle transduction and distribu-
tion to liver. Efficient muscle transduction occurred at high dose of
vector, independent of concentration, although this was not the
case when the overall dose of vector was decreased, at which point
more concentrated injections achieved higher muscle transduc-
tion. The highest concentration of injected vector in our study was
561012 GC/ml in mice and 361012 GC/ml in rhesus macaques,
which is essentially identical to the concentration of vector that
was injected in several AAV IM clinical studies including
hemophilia B, AAT deficiency, and the commercially approved
product Glybera [6,7,11,48]. In these studies, research subjects
received up to 60–100 injections at the highest dose. Previously,
attempts to increase concentration in order to decrease the
number of injections were hampered by the limit of AAV solubility
for most capsids in standard formulations being thought to be
1013 GC/ml. However, recently it has been reported that AAV8
vectors can be concentrated up to 1014 GC/ml [49]. Additionally,
low vector dose per site was reported to reduce the potential for
transgene-specific neutralizing antibodies following IM adminis-
tration of AAV vectors expressing cFIX in dogs [4,5]. These
studies had utilized AAV1 and AAV2 vectors and their differences
in hepatotropism versus AAV8 could provide explanation for the
lack of evidence for this effect with AAV8.
Our studies indicate that expression does not suffer, and in fact
may be improved, with higher concentrations of injected vector,
which suggests that more efficient transduction through capsid
engineering and/or optimized expression cassettes will likely yield
more production of the secreted transgene product. There clearly
will be a limit to how much transgene product can be produced
from a muscle cell, at which time protein biogenesis pathways will
be saturated and immune responses may be enhanced. In
conclusion, these pre-clinical data for delivery of AAV via IM
administration should inform on the safe and efficient develop-
ment of future AAV gene therapy products.
Materials and Methods
AAV vector productionAll AAV vectors were produced by the Penn Vector Core at the
University of Pennsylvania as described previously [50]. Briefly,
plasmids expressing firefly luciferase (ffLuc) or antibody constructs
driven by the cytomegalovirus (CMV) or human thyroxine
binding globulin (TBG) promoter were packaged with the AAV8
viral capsid. ffLuc and 201Ig IA plasmids driven by the CMV
promoter containing microRNA target sites were produced by
insertion of six copies of either the miR-122 or miR-206 target sites
into the 39 UTR region. Target sites were synthesized separated by
restriction enzyme recognition sites, indicated by the upper case
letters. The microRNA target site sequences were as follows:
6xmiR-122; TCTAGAcaaacaccattgtcacactccaGGATCCcaaacac-
cattgtcacactccaCGTACGcaaacaccattgtcacactccaACGCGTcaaac-
accattgtcacactccaCACGTGcaaacaccattgtcacactccaGCATGCcaa-
acaccattgtcacactccaGCGGCCGC, 6xmiR-206; TCTAGAccaca-
cacttccttacattccaGGATCCccacacacttccttacattccaCGTACGccaca-
cacttccttacattccaACGCGTccacacacttccttacattccaCACGTGccaca-
cacttccttacattccaGCATGCccacacacttccttacattccaGCGGCCGC.
MiceMale C57BL/6 and RAG KO mice at 6–8 weeks of age were
purchased from Charles River Laboratories (Wilmington, MA,
USA) and The Jackson Laboratory (Bar Harbor, ME, USA),
respectively. Mice were housed under specific pathogen-free
conditions at the University of Pennsylvania’s Translational
Research Laboratories. All animal procedures and protocols were
approved by the Institutional Animal Care and Use Committee
(IACUC, approval number 803395) of the University of
Pennsylvania. Mice were sacrificed by carbon dioxide asphyxia-
tion and death was confirmed by cervical dislocation.
Mice were anaesthetized with a mixture of 70 mg/kg of body
weight ketamine and 7 mg/kg of body weight xylazine by
intraperitoneal (IP) injection for all IM injections. Vector was
diluted in phosphate buffered saline (PBS) and IM injections were
performed using a Hamilton syringe. IV injections of vector were
performed by injection of 100 ml vector dilution into the tail vein.
Serum was collected weekly from mice administered with vectors
expressing secreted proteins by retro-orbital bleeds into serum
collection tubes.
NHPRhesus macaques (Chinese origin and captive bred, 3.75–
4.15 kg) were housed at the Nonhuman Primate Research
Program facility of the Gene Therapy Program of the University
of Pennsylvania (Philadelphia, PA) during the studies. Studies were
performed according to a study protocol approved by the IACUC
(approval number 804625), the Environmental Health and
Radiation Safety Office and the Institutional Biosafety Committee
of the University of Pennsylvania. All animals were housed in
stainless steel caging with perches and maintained on a 12-hour
light/dark cycle controlled via an Edstrom Watchdog system.
Temperature was maintained within the range of 18–26uC with
50% (610%) humidity. Animals were fed Certified Primate Diet
5048 (PMI Feeds Inc., Brentwood, MO, USA) two times per day
(morning and evening). Water was available ad libitum from an
automatic watering system. Food enrichment such as fruits,
vegetables, nuts and cereals were provided daily. Manipulanda
such as kongs, mirrors, puzzle feeder and raisin balls were
provided daily. Animals also received visual enrichment along with
human interaction on a daily basis.
All macaques had NAb titers of ,1:5 at the start of the studies
determined as described previously [51]. Prior to vector admin-
istration, macaques were anesthetized with a mixture of ketamine
(10–15 mg/kg) and dexmedetomidine (0.05–0.10 mg/kg) injected
IM into the bicep muscle. One macaque was administered with a
dose of 361012 GC/kg AAV8.CMV.201Ig IA into the vastus
lateralis muscle of both right and left legs as 1 ml injections per kg
body weight (vector concentration of 361012 GC/ml) for the
vector biodistribution study. To study the influence of vector dose
AAV8 Hepatic Targeting following IM Injection
PLOS ONE | www.plosone.org 8 November 2014 | Volume 9 | Issue 11 | e112268
per injection site, rhesus macaques were administered with a dose
of 361011 GC/kg as 1 ml injections per kg body weight (vector
concentration of 361011 GC/ml) into the vastus lateralis muscle
of both right and left legs or administered with the same vector
dose as 0.1 ml injection per kg body weight (vector concentration
of 361012 GC/ml) into the right vastus lateralis muscle. Blood
samples were taken pre-study and weekly during the study via
venipuncture of the femoral vein. Clinical pathology was
conducted by Antech Diagnostics (Irvine, CA, USA), including
complete blood counts and differentials, and complete clinical
chemistries, and transgene expression levels were measured in
serum by ELISA.
On day 90 post-vector administration the rhesus macaque that
received a dose of 361012 GC/kg was euthanized. The animal
was first anesthetized as described previously and euthanized using
sodium pentobarbital (80 mg/kg) injected IV. Death was con-
firmed by absence of heartbeat and respiration.
ImagingffLuc expression was visualized by whole-body bioluminescence
imaging weekly. Mice were injected IP with 10 ml/g body weight
of 15 mg/ml luciferin (XenoLight D-Luciferin Potassium Salt;
Perkin Elmer, Waltham, MA, USA). Five minutes post-luciferin
injection, mice were anaesthetized with ketamine/xylazine and 15
minutes post-luciferin injection mice were imaged using IVIS
Xenogen imaging system (Perkin Elmer, Waltham, MA, USA).
ffLuc expression was quantified using regions of interests in Living
Image 3.0 Software (Perkin Elmer, Waltham, MA, USA) and
measured in photons per second (p/s).
ffLuc Tissue AssayTotal injected muscle and a sample of liver were taken from
mice at the time of necropsy. Tissue samples were frozen on dry
ice and stored at 280uC. On the day of assay, tissue was weighed
and then homogenized in 500 ml 1x Luciferase Cell Culture Lysis
Reagent (CCLR; Promega, Madison, WI, USA) using the
QIAGEN TissueLyser (Valencia, CA, USA). Samples were then
centrifuged for 3 min at 10,000 x g at 4uC. The supernatant was
assayed following the Luciferase Assay System Protocol (Promega,
Madison, WI, USA). ffLuc tissue assay values were normalized to
total protein of the origin organ, either muscle or liver. A BCA
assay was performed on all supernatants from the ffLuc tissue
assay, diluted 1:1000 in PBS and using the Pierce BCA Protein
Assay Kit (Thermo Scientific, Waltham, MA, USA). Total protein
values were determined by the following calculation: (protein
concentration of tissue sample by BCA/weight of tissue sample
homogenized) x average weight of whole organ.
Detection of secreted proteins in mouse serumAntibody constructs were measured by ELISA where all
reagents were from Sigma-Aldrich (St. Louis, MO, USA) unless
otherwise stated. ELISA plates were coated with 5 mg/ml protein
A, diluted in PBS and incubated overnight at 4uC. Wells were
washed eight times with 0.05% Tween 20 in PBS and blocked with
1% BSA in PBS for one hour at room temperature. Following
removal of the blocking buffer, heat inactivated serum samples
diluted in PBS were added to the plates and incubated at 37uC for
one hour. Plates were then washed eight times, blocked as
described previously and Bio-SP-conjugated Affinipures Goat
Anti-Human IgG antibody (Jackson ImmunoResearch Laborato-
ries, Inc., West Grove, PA, USA) was added at a 1:10,000 dilution.
Following incubation at room temperature for one hour, plates
were washed eight times and strepdavidin protein conjugated to
horseradish peroxidase (HRP) was added at a 1:30,000 dilution.
After another incubation at room temperature for one hour, plates
were washed eight times and 3,39,5,59-tetramethylbenzidine
(TMB) was added for detection. The reaction was stopped after
30 minutes at room temperature using 2N sulfuric acid and plates
were read at 450 nm using a BioTek mQuant plate reader
(Winooski, VT, USA).
Detection of secreted proteins in NHP serumDetection of 201Ig IA in NHP serum was measured by antigen-
specific capture ELISA. Following a similar procedure as that
described above, ELISA plates were coated with 2 mg/ml
SIVmac251 gp120 protein diluted in PBS and incubated
overnight at 4uC. Wells were washed five times and blocked with
1 mM EDTA, 5% heat inactivated PBS, 0.07% Tween 20 in PBS
for one hour at room temperature. All subsequent steps were the
same as described previously.
LacZ stainingLacZ gene expression was examined 21 days after vector
administration by methods described previously [52].
Vector biodistributionLiver and muscle samples were snap frozen at the time of
necropsy and DNA was extracted using the QIAamp DNA Mini
Kit (Qiagen, Valencia, CA, USA). Detection and quantification of
vector genomes copies (GC) in extracted DNA were performed by
real-time PCR as described previously [53]. Briefly, genomic DNA
was isolated and vector GCs were quantified using primers/probe
designed against the SV40 poly adenylation sequence of the
vector. Quantification of GC from liver was performed on one
liver sample from each mouse (n = 4/group) and on a sample from
each of the lobes of the liver in the NHP, data is presented as an
average of the lobes. DNA extraction was performed on a
homogenate of the entire gastrocnemius muscle from mice or from
samples taken at 12 sampling sites throughout the injected vastus
lateralis muscles and control bicep muscle from NHP. The average
of the 12 samples per muscle is presented.
RNA isolation and real-time PCRRNA was isolated from NHP muscle and liver samples using
TRIZOL (Life Technologies, Carlsbad, CA, USA) according to
the manufacturer’s protocol. 10 mg of RNA was then treated with
DNase I (Roche, Basel, Switzerland) according to the manufac-
turer’s protocol. The RNeasy Mini Kit (Qiagen, Valencia, CA,
USA) was used to remove DNase prior to cDNA synthesis by
reverse transcription using the Applied Biosystems High Capacity
cDNA Reverse Transcriptase Kit (Life Technologies, Carlsbad,
CA, USA). Real-time PCR was then performed on cDNA with
primers binding to the 201Ig IA transgene with Power SYBR
Master Mix for detection or primer/probe set for 18S with
TaqMan Gene Expression Master Mix for detection (Life
Technologies, Carlsbad, CA, USA). Relative transcript expression
was determined using the DDCT of each sample normalized to
18S expression.
Statistical analysisAll analyses were performed in Prism (GraphPad Software, San
Diego, CA, USA). A p value of ,0.05 was considered significant.
Comparisons between two groups were performed using unpaired
Student’s t-test and comparisons between multiple groups were
performed using one-way analysis of variance (ANOVA, Tukey’s
Multiple Comparison post-test). All values expressed as mean 6
standard error of the mean (SEM).
AAV8 Hepatic Targeting following IM Injection
PLOS ONE | www.plosone.org 9 November 2014 | Volume 9 | Issue 11 | e112268
Acknowledgments
We would like to thank Deirdre McMenamin, Christine Draper and Erin
Bote (University of Pennsylvania Perelman School of Medicine, Gene
Therapy Program) for invaluable technical assistance.
Author Contributions
Conceived and designed the experiments: JAG APT JMW. Performed the
experiments: JAG HP JO CAM-J OA AM SC PB. Analyzed the data: JAG
PB JMW. Contributed reagents/materials/analysis tools: RLG SR SC PB
APT. Wrote the paper: JAG APT JMW.
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